Mineral and heavy metal content in dry dog foods with different main animal components

Dog caregivers, mainly for economic reasons and easy availability, choose dry, over the counter diets (OTC). The mineral composition of OTC foods depends primarily on the components used in the production of the pet food. Regardless of the main component of the food, it must meet the recommended minimum mineral content, established by nutritional guidelines. Therefore, the aim of this study was to determine the mineral (Ca, K, Mg, Na, Fe, Mn, Zn, Cu, Mo) and heavy metal content (Pb, Co, Cd, Cr, Ni) using the methods of colorimetry and mass spectrometry, of OTC dry dog foods and to compare with the FEDIAF and AAFCO nutritional guidelines. Dry foods pose no risk to dogs in terms of heavy metal content. The worst results in terms of mineral content were obtained in mixed foods, therefore it is worth considering feeding the dog a mono-protein food. The PCA analysis disproved our hypothesis and revealed that the main animal source did not statistically significantly affect the levels of minerals and their ratios. However, the analysis of contrasts confirms the differentiation of the content of individual minerals between the groups of foods. For the first time, we proved that pet food with a mineral composition similar to the MIN-RL may be characterized by unfavorable mineral ratios.

Trace elements and heavy metals. FEDIAF provides both the MIN-RL (3.60 g/100 g DM) and the MAX-LL (68.18 mg/100 g DM) of iron (Fe), while AAFCO only recommends the minimum level (4.0 mg/100 g DM) ( Table 3). All analyzed foods met the MIN-RL for iron, without exceeding the legal limit (MAX-LL). Statistically the highest amounts of iron were found in the M4 food (46.62 mg/100 g DM), while the lowest was determined in food with fish (in the F8 food-4.82 mg/100 g DM). Statistically the highest amounts of iron were found in the group of mixed foods (23.67 mg/100 g DM) (Table 4), while the lowest-in the group of other foods (11.93 mg/100 g DM). All groups of foods differed significantly in terms of iron content. In the case of recommendations regarding the level of zinc (Zn), a similar situation can be observed-FEDIAF provides the MIN-RL (7.20 mg/100 g of DM) and the MAX-LL (22.70 mg/100 g of DM), while AAFCO-only the minimum recommended level (8.0 mg/100 g of DM). The lowest level of zinc was found in the O3 food (2.53 mg/100 g DM). The significantly lowest average zinc level was found in the group of other foods (5.53 mg/100 g DM), and the highest-in the group of foods with fish (14.10 mg/100 g DM). All groups of foods differed significantly in terms of zinc content. Fourteen foods did not meet the FEDIAF and AAFCO minimum recommended levels of zinc, and two foods (F1 and M2) exceeded the legal limit of this trace element (78.95 and 24.92 mg/100 g DM, respectively). The recommended minimum content of manganese (Mn) by FEDIAF is 0.58 mg/100 g DM, and by AAFCO-0.50 mg/100 g DM. The legal limit is specified only by FEDIAF-17.00 mg/100 g DM. Two of the analyzed foods did not meet the recommended minimum level (F8-0.45 and M2-0.54 mg/100 g DM). The highest level of Mn was found in the O4 food (7.56 mg/100 g DM). All groups of foods differed significantly in terms of manganese content. In the case of copper (Cu), the minimum recommended level in dog food is 0.72 and 0.73 mg/100 g DM according to FEDIAF and AAFCO, respectively. The legal limit is 2.80 mg/100 g DM. The significantly highest average content of copper was found in the group of foods with beef (1.84 mg/100 g DM), while the lowest-in the group of other foods (0.88 mg/100 g DM). All groups of foods differed statistically significantly in terms of copper content. Some of the foods did not meet the minimum recommended levels-F8 (0.22 mg/100 g DM), O2 (0.24 mg/100 g DM and O3 (0.16 mg/100 g DM). One food (B1) exceeded the MAX-LL (2.94 mg/100 g DM). The nutritional guidelines do not state limits for molybdenum (Mo), however, its presence was found in all foods at an average level of 0.10-0.11 mg/100 g DM. The analyzes did not detect the presence of cobalt (Co), cadmium (Cd), chromium (Cr) and nickel (Ni) in the foods, while the presence of lead (Pb) was found in five foods (Table 3)-one with fish, two with poultry and two mixed (F1, P1, P2, M1, M5). Table 1. Content of macrominerals (g/100 g DM) in analyzed dry dog foods. DM, dry matter; MIN-RL, minimum recommended level; MAX-RL, nutritional maximum limit; F, foods with fish; P, foods with poultry; L, foods with lamb; B, foods with beef; M, mixed foods; O, other foods; means with at least one same letter in the superscript (a, b, c, …) not differ statistically at P = 0.05 (for all columns separately). * No data. Comparative analysis. PCA analysis based on the mineral composition of the dry dog foods indicated that the first two components accounted for 47.13 percent of the total variance (Fig. 1A). The analysis allowed the distribution of the foods in the quadrants of the 2 factor coordinate plot for the cases (Fig. 1B). The foods that make up single-element sets in quadrant I are F1 and F7. These foods are distinguished by a particularly high content of K and Zn, but also all foods in this quarter were characterized by an increased content of Cu and Na. Quarter IV foods contain a higher content of Mn, Mg, Fe and Ca than other foods. Foods forming a separate group in this quarter, with a particularly high content of the above-mentioned elements, are foods F12, O4 and L3. It is worth emphasizing that this quarter contains the vast majority of foods with lamb. Quarter III includes the FEDIAF 10 guidelines and forms a group with F8 food, which suggests a close but not complete compliance of its mineral composition with the MIN-RL, which confirms that it does not meet the requirements for Mn and Cu. Generally, foods in this quarter have a higher content of phosphorus than in other foods. In quarter II,

Contrasts Na
Item www.nature.com/scientificreports/ a separate group is formed by O2 and O3 foods, which have in common the low content of minerals, especially Mn, Mg, Fe and Ca (Fig. 1A). The PCA analysis carried out on the ratios of elements showed that the source of animal raw materials did not significantly affect the ratios of elements in the tested foods, because in the coordinate system there were no uniform groups of foods determined on the basis of the main component ( Fig. 2A, B). For example, foods from the others group, which are in different quarters, such as food O1 (quadrant II) and www.nature.com/scientificreports/ food O3 (quadrant IV). The ratios of the content of elements in the absolute majority of pet foods are far from those resulting from the FEDIAF. The presence of O3, O2 and F8 foods on the market should be emphasized, the ratios of which were extremely unfavorable, which should raise doubts as to the safety of their long-term administration to animals. These foods were characterized by narrow Ca:P, Ca:Mg, and Fe:Cu ratios and a wide Cu:Zn ratio. The conducted analysis also allows to indicate one food (M2) whose mineral content ratios are close to the ratios resulting from FEDIAF. It is worth emphasizing that the food with a similar composition to MIN-RL was the F8 food, but the food with the most similar ratios was the M2.

Discussion
A complete and balanced food is essential for the health and well-being of dogs. Adequate diets at every stage of life provide the nutrients needed for reproduction, growth and a long, healthy, active adult life. They also prevent nutritional disorders that can occur due to nutritional deficiencies or excesses. Both the FEDIAF and AAFCO nutritional guidelines establish minimum recommended levels for macrominerals (Ca, P, K, Na, Mg, Cl) and trace elements (Cu, I, Fe, Mn, Se, Zn), however, the FEDIAF guidelines also provide a maximum level of a nutrient in a complete pet food and legal limit for most of them (MAX-RL-Ca, P; MAX-LL-Cu, I, Fe, Mn, Se, Zn). The legal limit applies only when a specific element is included in the recipe as an additive, but it applies to the total amount present in the finished product, and if it comes solely from the ingredient, it does not apply. The legal limit is mandatory and always applies to all life stages. The nutritional maximum is the highest level that is not supposed to cause any harmful effect. Unless the life stage is indicated it applies to all life stages 10 . The mineral content found will be compared to both MAX-RL (macrominerals) and MAX-LL (trace elements), in order to show irregularities in the balance of foods in terms of mineral content. In our research, the level of calcium was Table 4. Contrasts between trace elements in the analyzed groups of dog food. F, foods with fish; P, foods with poultry; L, foods with lamb; B, foods with beef; M, mixed foods; O, other foods.

Contrasts Fe
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Contrasts Mn
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Contrasts Zn
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Contrasts Mo
Item  13 in adult dogs shown that excess dietary calcium does not lead to adverse health effects, which means that a deficiency in the diet should be of more concern. Secondary hyperparathyroidism (NSHP), osteodystrophy and osteopenia are common complications that can occur in animals consuming an unbalanced diet. The root cause of NSHP is calcium deficiency, which can occur due to an inability to absorb Ca, a lack of  14 . By definition, a complete food should be balanced in such a way as to meet the MIN-RLs for all minerals. However, as our research shows, additional supplementation with calcium-rich products may be necessary in some cases. In a dog's diet, the sources of calcium can be primarily bones or eggshells. According to the research of Ebeledike et al. 15 the best source of calcium among animal products is chicken meat and offal, followed by goat, cattle and pig. In our study, dog foods with lamb were best in terms of calcium content, containing 0.62 g/100 g of DM calcium. In the studies of Pereira et al. 16 , the average calcium level in adult dog food was 1.94 g/100 g DM, so it was almost three times higher than the levels found in our studies. Despite this, it was within the recommended levels, not exceeding MAX-RL. Sgorlon et al. 17 found an average calcium content of extruded dry foods of 1.08 g/100 g DM, which was also higher than the levels determined in our study. Phosphorus has a major role in energy metabolism as a component of adenosine triphosphate 18 . Importantly, the consumption of diets rich   19 . This is especially dangerous for dogs suffering from kidney disease. In addition, wet foods have been shown to pose a risk in this regard as they contain much higher levels than the FEDIAF recommended MIN-RL, exceeding the MAX-RL 20 . According to studies 15,21,22 , the content of phosphorus in the meat of individual animal species can be ranked as follows: fish > beef > lamb > poultry > pork. The largest amounts are contained in the bone elements of these animal species 15 . Long-term deficiency of phosphorus in the diet leads to hypophosphatemia, anorexia, disturbances in appetite and growth 23 . Elevated serum phosphate levels are a recognized risk factor for cardiovascular disease and mortality in chronic kidney disease 23 . As shown by Dobenecker et al. 2 the use of certain inorganic phosphates in pet food is potentially harmful and should be limited. In our research, the highest average level of phosphorus was found in foods with lamb (1.36 g/100 g DM) and fish (1.35 g/100 g DM). The overall average of all foods was 1.28 g/100 g DM. The results found are higher than those obtained by Sgorlon et al. 17 (1.0 g/100 g DM) and   www.nature.com/scientificreports/ Pereira et al. 16 (1.17 g/100 g DM). Potassium aids in the functioning of electrical charges in the heart, nerves, and muscles. Sodium aids in the production and transmission of nerve impulses. In dog diet good sources of sodium can be eggs 24 . Meat and fish are good sources as well, but processed ones contain more sodium 25 . This means that dogs fed with "human" food and getting leftovers are exposed to higher sodium doses. In our study, the highest level of potassium was found in group of other foods (0.97 g/100 g DM) and foods with poultry (0.94 g/100 g DM). In the studies of Pereira et al. 16 , an average of 0.77 g/100 g DM potassium was found, and in Sgorlon et al. 17 -0.77 g/100 g DM. In case of sodium, its highest amounts were found in group of other foods (0.68 g/100 g DM) and foods with poultry (0.42 g/100 g DM). The overall average of all foods was 0.50 g/100 g DM. In the studies of Pereira et al. 16 , an average of 0.69 g/100 g DM sodium were found and was higher than in our research. However, Sgorlon et al. 17 found lower sodium content in dog foods-at the level of 0.32 g/100 g DM. Magnesium is important in many biological functions as a coenzyme in the body of both humans and animals 26 . Due to the proliferative nature of blood cells, the microenvironment which regulates the latency of the hematopoietic stem cells are key to the survival of animals in health and disease 27 . Magnesium disorders have been associated with prolonged hospitalization and higher mortality rates, with neurological and cardiovascular clinical signs 28 . In our study, the highest amount of magnesium was found in foods with lamb (0.23 g/100 g DM). In study by Pereira et al. 16 magnesium content was determined at level of 0.15 g/100 g DM which was in line with our results for poultry, beef, mix and other foods. Sgorlon et al. 17 obtained lower results-at an average level of 0.10 g/100 g DM. The nutritional guidelines provide pet food manufacturers nutritional recommendations to ensure the production of a balanced and nutritionally healthy pet food, and is periodically updated based on the latest scientific publications. An example of a change that has occurred to the FEDIAF nutritional guidelines is the legal limit for iron in dry dog food. In the 2019, it was 142 mg/100 g DM for food, while in the latest guidelines it was reduced to 68.18 mg/100 g DM. Because, in dogs, the centrilobular hepatic region is where copper accumulates, colocalization of copper and toxic metabolites or electrophiles magnifies the risk of regional cellular injury. Of additional concern is that centrilobular regions are the last zone to extract oxygen from sinusoidal blood. Consequently, these hepatocytes have a heightened risk for ischemic, hypotensive, or hypoxia-related oxidative injury. Thus, colliding factors may initiate sudden hepatocyte injury in dogs with substantial centrilobular copper accumulation. Examples of this so-called "2-hit phenomenon" include sudden dramatic increases in serum alanine transaminase activity in combination with severe hemolytic or blood loss anemia, hypoxemia, cardiac failure, hypotensive shock, gastric dilatation volvulus, hypotension during general anesthesia, and administration of cytochrome P450-metabolized xenobiotics (e.g., NSAIDs) that form oxidative adducts or electrophiles 29 . The greatest amount of iron in animal products can be found in lamb and beef compared to chicken and pork 9 . The excess iron acts as a poison in dog, and causes damage to the gastrointestinal, liver, metabolic, nervous, and cardiovascular systems. Iron is used by nearly all cells in the body and is an important component of many processes, including erythropoiesis 30 . In veterinary medicine, dietary iron deficiency in adult dogs is rarely seen 31 . In our research, mixed foods were the richest in terms of iron content (23.67 mg/100 g DM). In the studies of Sgorlon et al. 17 found an average iron level of 16.54 mg/100 g DM, which was similar to the value we found in our research in foods with poultry (16.83 mg/100 g DM). An excess of iron in the diet can lead to mild gastrointestinal damage. High levels of copper interfere with iron absorption, which reduces its use by the body. This can lead to anemia and frequent diarrhea 32 . In our study, the level of copper in all foods was lower than the level of iron, which resulted in low ratios of these elements, similar to the studies of Pereira et al. 16 and Sgorlon et al. 17 . Copper is involved in erythropoiesis and supports iron absorption. Participates in pigmentation of the skin and hair. Deficiency, although rare, can cause muscle dysfunction. In the liver, it can cause acute hepatitis with immediate consequences, or it can cause chronic damage over time, leading to extensive scarring (cirrhosis) and liver failure. Excessive accumulation of copper in the liver as a cause of hepatitis and cirrhosis was first demonstrated in Bedlington terriers. Copper excess can be found in dogs with normal liver histology, dogs with hepatitis, and dogs with end-stage cirrhosis. A growing body of evidence suggests that copper is a cause of liver disease in non-Bedlington dog breeds 33 . Excess copper causes damage to hepatocytes 29 . Among slaughter animals, the richest source of copper is lamb 9 , therefore it is assumed that lamb foods should contain the most of it, and therefore should be used with caution in the nutrition of dogs with liver problems. Our research does not confirm that foods with lamb are supposed to be a good source of copper. Its highest levels were found in group of foods with beef (1.84 mg/100 g DM) and lamb (1.51 mg/100 g DM), which was similar to the studies by Sgorlon et al. 17 (1.50 mg/100 g DM). On the other hand, Pereira et al. 16 found a 70 percent higher average copper level of 2.28 mg/100 g DM. Zinc is important for cellular immunity, reproductive and skin function, and wound healing 34 . The best source of zinc is beef 9 . In our research, the highest average level of zinc was found in the group of foods with fish (14.10 mg/100 g DM), because the F1 food almost four times exceeded the MIN-RL, which influenced the increase in the average obtained in this group of foods. There is a risk that the producer could have used a different raw material for the production, not declared on the label, which resulted in a very high level of zinc in this pet food. The vegetable component in this food was, among others, chickpeas, which are a rich source of zinc. The obtained result may therefore be a reflection of the component composition. It may also be related to the accumulation of zinc in the raw material, depending on the feed consumed by the fish or on the mineral supplements used in the F1 food production. In Sgorlon et al. 17 study, the average level of this element in extruded dry dog foods was 23.15 mg/100 g DM, while in study by Pereira et al. 16 32.50 mg/100 g of DM. In a study by Gregório et al. 34 the average content of zinc in dry foods was about 29.52 g/100 g DM. Importantly, most of the foods had Zn levels higher than the established limit (22.7 mg /100 g DM). High levels of copper in the diet and low levels of zinc are significantly associated with high levels of copper in the liver. Dietary copper and zinc at current levels in commercially available dry dog food may affect copper in the liver and may be a risk factor for the development of copper-associated hepatitis in Labrador Retrievers with a genetic susceptibility to copper 35 . In our study, the average level of copper in all food groups was ten times lower than that of zinc, as in Pereira et al. 16 and Sgorlon et al. 17  www.nature.com/scientificreports/ collagen, which gives strength to soft tissues. According to the research of Gerber et al. 9 from meat, lamb is the best source of manganese in comparison with foal, chicken, beef, pork. Compared to the literature data, the level of manganese in our study was relatively low. The highest was found in the group of foods with lamb (3.57 mg/100 g DM), and compared to the average results obtained by Pereira et al. 16 (7.98 mg/100 g DM) and Sgorlon et al. 17 (10.66 mg/100 g DM) the level was quite low. As a result of natural processes and human activity, molybdenum enters soil and water, and then enters the food chain through plants and livestock. Normally pure molybdenum is not added to pet food, but ordinary ingredients carry this element. Experiments in rats have shown that molybdenum is an essential nutrient, while excessive consumption is toxic 36 . These characteristics of molybdenum most likely apply to dogs and cats. The toxicity of high molybdenum intake animals is related to the induction of secondary copper deficiency; the condition can be cured or prevented with copper supplementation 37 . Among slaughter animals, the best source of molybdenum is skinless chicken breast in comparison with foal, lamb, beef, pork 9 . In our study, the amounts of molybdenum found were relatively similar in all food groups (average 0.100-0.110 mg/100 g DM), but higher than those found by Pereira et al. 16 (0.049 mg/100 g DM). In order to increase the proportion of minerals in the diet, vitamin-mineral supplements 38 can be used, which is common practice among dog caregivers on a home or raw diet. Most vitamin and mineral supplements, in the amounts recommended by manufacturers, do not guarantee the minimum nutritional recommendations for the following elements: calcium, potassium, magnesium, sodium, phosphorus, selenium and zinc. In study by Zafalon et al. 38 most vitamin and mineral supplements have been found to fall short of minimum recommendations for the most important minerals, and when formulated by untrained professionals, even with supplementation, homemade food may still be deficient in nutrients. Moreover, some analyzed vitamin and mineral supplements may suggest a risk of mercury poisoning in domestic animals 38 . Our research did not detect the presence of Co, Cr, Ni. However, they can be present in dog food 16,17 . In the study by Pereira et al. 16 , the content of Co, Cr, Ni in dog foods was at the level of 0.014, 0.076, 0.081 mg/100 g DM, respectively. On the other hand, in study by Sgorlon et al. 17 the level of these elements was 0.025, 0.153, 0.093 mg/100 g DM. These data indicate that although these elements are present in small amounts in food, dogs are still exposed to them due to the possibility of accumulation. A type of threat are heavy metals, the sources of which in food chains are pollution, soil, machinery and water used in production. The most dangerous heavy metals unnecessary for the body, include cadmium (Cd), lead (Pb), mercury (Hg) and arsenic (As). Their impact on the dog body is varied and depends on the amount of the absorbed dose, duration of the contamination process and the age of the animal. These elements have the highest accumulation factor. They are involved in the development of diseases of the nervous, skeletal and blood systems. They also have a carcinogenic effect, contributing to the development of neoplasms. The maximum allowable levels (MAL) of heavy metals in foodstuffs are regulated by the Commission Regulation (EC) establishing the MAL of certain contaminants in foodstuffs, but there is no regulation specifying the level of these contaminants in pet food. According to Directive 2002/32/EC 39 , referred to in the Code of Good Manufacturing Practice 40 and the subsequent document amending them 41 , the level of lead in complete feed (i.e. within the meaning of the legal act-also in complete dog food) should not exceed the maximum content of 5 mg/kg in a corresponding feed with a moisture content of 12 percent. Moreover, in the case of food products, in 2021 the EU lowered the limits for heavy metals-cadmium and lead 42,43 . The source of heavy metals can be pet food ingredients (vegetables, fruits, and animal products). Pork fat had almost three times higher concentrations of arsenic, than fish oil and poultry fat. Moreover, pork fat compared to fish oil and poultry fat, was the only one containing mercury 44 . Other studies indicate that foods with fish as the main meat component, which contain higher amounts of heavy metals than foods based on chicken or red meat 4 . Our research analyzed the levels of cadmium and lead. Cadmium was not found in any of the foods, while lead was present in one fish, two poultry and two mixed foods. The highest level found was for food with fish-0.046 mg/100 g DM. In the study of Sgorlon et al. 17 the average level of cadmium and lead found was 0.030 and 0.307 mg/100 g DM, while in Pereira et al. 16 0.015 and 0.018 mg/100 g DM. It has been shown that dry food has a higher concentration of most heavy metals than wet food. Although the amounts found are not high, it should be borne in mind that chronic exposure to their presence has negative health consequences, however, levels of chronic exposure to toxicity are very unlikely. In a study by Kim et al. 4 , fish-based foods had significantly higher arsenic, cadmium, and mercury content than poultry or red meat-based diets. Red meat-based diets (beef, venison, and bison) were characterized by a higher concentration of lead than poultry and fish diets. Based on these results, it can be concluded that commercial dog food appears safe for chronic consumption, and the concentration of heavy metals depends on the primary protein sources 4 . While our research has focused on the mineral content of foods depending on the animal raw material, it is worth bearing in mind that sometimes the composition of the pet food is balanced in such a way that it is impossible to naturally provide all the minerals in amounts that meet the MIN-RL. Therefore, manufacturers use permitted mineral dietary supplements to increase the level of a given mineral in dog food, in the form of sulfates, carbonates and other compounds. However, the safety of such practices is questioned, as underestimating the proportion of "artificial" additives-which will be discussed later-may adversely affect the health of the animal, therefore it is necessary to monitor the composition of foods and establish safe levels of dietary additives, including minerals. An example of a mineral whose differences in bioavailability should be analyzed is ferrous carbonate, which is generally less bioavailable than ferrous sulfate. While iron carbonate may be effective for adult animals, it would be insufficiently bioavailable for young animals which require a highly effective iron source for a rapid response in hemoglobin synthesis 45 . Given the limited information on iron toxicity in dogs and potential differences between breeds in trace element sensitivity, it is imperative to analyze the exact composition of complete feeds not only for adult animals, but also for young ones, which should be the subject of our next research. Because of the close relationship of calcium and phosphorus, AAFCO and FEDIAF guidelines provide a minimum calcium-to-phosphorus ratio of 1:1 and a maximum ratio of 2:1. In our study, the best balanced foods in terms of calcium to phosphorus ratio were foods with lamb, while the worst in this respect were mixed foods, with at least 2 different species as the main animal www.nature.com/scientificreports/ components. In one mixed food, the ratio was 0.06:1, which is undoubtedly a very disturbing result. In the study of Pereira et al. 16 the Ca:P ratio was 1.64:1, while in the study by Sgorlon et al. 17 − 1.1:1, these results do not confirm ours. Importantly, a clinical study found that any growing dog that did not receive the appropriate food and supplements depending on age and breed showed joint thickening, long bone inversion, fore and hind limb inversion, limb unfolding, and difficulty moving 46 . It is important because the caregivers of dogs growing after the age of 1 year often switch from puppy food to maintenance food for adult dogs, despite the fact that dogs of large breeds grow slower and longer need appropriate food for growing dogs, not only with the appropriate proximate composition, but also with an appropriate content of minerals, including the essential calcium and phosphorus, which are involved in the development of the skeleton. A reduced Na:K ratio is a common symptom in patients with adrenal insufficiency, and adrenal insufficiency is considered one of the more likely diagnoses in dogs with a low Na:K ratio 47 . Sgorlon et al. 17 found potassium and sodium content in dog foods at the level of 0.46 and 0.32 g/100 g DM, respectively, which gives Na:K ratios 0.89:1 and 0.70:1, respectively. In our research, the ratio of 0.70:1 was found in the group of other foods. The lowest ratio was found in the group of foods with beef (0.49:1). The action of magnesium in the body is suppressed by excess calcium, which may contribute to calcium deposition in the urinary tract and gallbladder. Highest ratio of calcium to magnesium may has a certain relationship with the incidence rate of urolithiasis in dog. In our research, the ratio of these elements ranged from 1:0.30 in foods with beef to 1:0.46 in those with fish. In the studies of Pereira et al. 16 , this ratio was 1:0.13, while in Sgorlon et al. 17 1:0.10. An important issue that cannot be overlooked in the context of the mineral composition of complete dog foods is the content of additives. Many additives are used in dog food to increase the taste of the food, extend its freshness or have a positive effect on the animal's body, for example by improving the functioning of joints or enriching the intestinal microflora. All additives must be approved for use and administered in the correct amounts 48 . In the countries belonging to the European Union, the matter of additives used in animal nutrition is regulated by the Regulation of the European Parliament and of the Council 49 . Depending on their properties and functions, additives have been divided into the following categories 49 : (1) technological additives-substances that prolong the freshness of food and are responsible for its structure, including preservatives, antioxidants, emulsifiers, stabilizers; (2) sensory additives-dyes and preparations aimed at improving the taste and smell of food; (3) nutritional additives-positively affecting the functioning of the body, for example vitamins, minerals and amino acids; (4) zootechnical additives-substances that increase digestibility and have a positive effect on the intestinal microflora; (5) coccidiostats and histomonostats-substances limiting and inhibiting the development of protozoa.
Mineral substances added to pet food therefore belong to point 3-nutritional additives. However, adding them can be problematic, because it can lead to their excess in the diet. As shown by Kazimierska et al. 50 , for example, Zn in dog foods was more likely to be in excess than in deficit. Interestingly, dog foods exceeding the maximum legal limit of Zn, Cu and Fe were fortified with nutritional additives, although apparently they did not require any supplementation, as this led to exceeding the maximum legal limit. In the studies of these authors, it was also observed that dog foods without declared supplementation with nutritional additives met the minimum recommended levels of trace elements, and in two foods they even exceeded the acceptable level. Interestingly, according to the Code of Good Labelling Practice for Pet Food, produced by the European Pet Food Industry Federation, there is no obligation to declare additives with no legal maximum limit 48,51 .
Dry foods pose no risk to dogs in terms of heavy metal content. The worst results in term of mineral content were obtained in mixed foods, therefore it is worth considering feeding the dog a mono-protein food. When feeding dogs with extruded dry foods, supplementation with calcium may be necessary due to its deficiency in foods, especially in mixed foods, with components from at least two different types of animals. The PCA analysis disproved our hypothesis and revealed that the main animal source did not statistically significantly affect the levels of minerals and their ratios. What is more, pet food with a mineral composition similar to the MIN-RL may be characterized by unfavorable mineral ratios.

Methods
Material. Over the counter compete dry dog foods (41) for adult dogs were selected in packages from the range of 0.3 to 2.5 kg. These foods were randomly chosen at Polish pet stores and online stores. Dog foods were bought in January 2022. Foods were divided into groups depending on the main animal component: fish (F), poultry (P), lamb (L), beef (B). A group of mixed foods (M) was distinguished, which contained at least two different sources of animal components, and a group of other foods (O) with unconventional sources of animal protein (Table S1). All bags were stored in laboratory room temperature (approx. 18-21 °C) until analyzes. Bags were opened on the same day. From each bag, a representative sample was collected for laboratory analyzes 52 . Samples of foods were ground in a laboratory mill type KNIFETEC 1095 (Foss Tecator, Höganäs, Sweden), placed in sterile containers and marked with the symbols 1-41 (Tables 1, 2

Chemical analyzes.
To determine the dry matter, the samples were dried at 105 °C until constant weight according to AOAC 53 . The total content of potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), molybdenum (Mo), lead (Pb), cobalt (Co), cadmium (Cd), chromium (Cr) and nickel (Ni) was determined by wet mineralization in a mixture of nitric acid (V) and perchloric acid (VII) 52  www.nature.com/scientificreports/ individual elements was started with a standard curve taking into account the weight of the test portion and the dilutions used. The material for P concentration analyses was subjected to mineralization in concentrated sulfuric acid (H 2 SO 4 ) and perchloric acid (HClO 4 ). The phosphorus (P) content was determined by the Egner-Riehm colorimetric method, with ammonium molybdate on a Specol 221 apparatus spectrophotometer (Carl Zeiss Jena, Germany). The absorbance value of the sample, determined spectrophotometrically, from P 2 O 5 to total phosphorus, was calculated according to the chemical equivalent (0.436). The credibility of the method used has been confirmed by comparative studies, incl. calibration curve, using the calibration series method. Macrominerals content was expressed as g per 100 g DM of dog food, the content of trace elements and heavy metals was expressed as mg per 100 g DM of dog food. All chemical determinations were performed in triplicate and presented as mean values. The accuracy of the analytical methods was verified based on certified reference material skimmed milk powder (ERM®-BD151), which was obtained from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium).
Nutritional adequacy. The results of the analyzes are expressed in 100 g DM (Tables 1 and 3). The demand for nutrients recommended by the nutritional guidelines is expressed in a unit per 100 g DM, assuming an energy density of 4 kcal 10 . The levels of all nutrients were compared to minimum recommended level (MIN-RL), nutritional maximum limit (MAX-RL) and legal limit (MAX-LL) 10,11 . In the case of mineral ratios, the nutritional guidelines provide MIN-RL and MAX-RL for Ca and P only. That is why the mineral ratios were calculated on the basis of the recommended content of a given element in dog food.
Statistical analyzes. Statistical analyzes were carried out using the STATISTICA v13.3 software (TIBCO Software Inc., Palo Alto, CA, USA) 54 . The significance of differences between the means was assessed using the Tukey test at P = 0.05. The method of contrasts was used to compare the means for the groups of dog foods.

Data availability
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